Friedreich's ataxia (FRDA) is due to homozygous transmission of guanine-adenine-adenine (GAA) trinucleotide repeat expansions in parental frataxin (FXN) genes. The result is deficiency of frataxin. Frataxin is a mitochondrial protein that undergoes specific maturation during and after transfer into the mitochondrial interior. Its putative normal function is the biogenesis of iron- sulfur (Fe-S) clusters conveying iron homeostasis to the entire cell. An inadequate supply of Fe- S clusters seriously impairs the activities of complexes I, II, and III of the mitochondrial electron transport chain, and the citric acid cycle enzyme, aconitase. In addition to suboptimal biosynthesis of high-energy phosphates, lack of frataxin also heightens sensitivity to oxidative stress, presumed to be mediated by free or loosely bound iron. Tissue damage in FRDA is very diverse. In the heart, the disease causes limited accumulation of iron in cardiomyocytes but similar restricted iron excess has not been demonstrated in central or peripheral nervous systems. Assays of total iron do not identify the small catalytic amounts of the metal that are required for the generation of toxic oxygen species. It is also unlikely that human autopsy tissues are suitable for the direct determination of this small pool of highly reactive iron. Nevertheless, failing iron homeostasis can still be assessed by the "downstream" effects of iron on iron-responsive proteins among which ferritin, mitochondrial ferritin, and ferroportin are most likely to undergo changes. In FRDA, the cerebellar dentate nuclei and the dorsal root ganglia (DRG) of the spinal cord are highly vulnerable. This research will test the hypothesis that the adverse effect of frataxin deficiency on these anatomical sites is the result of diffuse or localized iron excess. Regional increase can be determined directly by a new technology, high-definition X-ray fluorescence (HDXRF);and indirectly by a systematic examination of iron-responsive proteins. The investigator will combine HDXRF with slide techniques and biochemical assays of ferritin and ferroportin. HDXRF "maps" iron in tissue blocks and allows its quantification based on standards. Sections of the same block displaying immunocytochemical reaction products of iron-responsive proteins and iron "maps" will be matched precisely to reach a correlation of iron and proteins. Levels of ferritin and ferroportin are expected to be inversely correlated with concentrations of frataxin. A correlation may also exist with the age of the patient at the time of death or the duration of his (her) disease. The expression of mitochondrial ferritin in response to frataxin deficiency is more complex, and results are expected to be all-or-none, as previously reported for FRDA heart. FRDA may also cause iron dysmetabolism due to incorrect intramitochondrial frataxin maturation. This potential contributing factor will be examined by Western blotting of frataxin precursors and the mature functional protein, and in a more robust approach, isotope-coded affinity tag technology and tandem mass spectrometry. This research also utilizes advanced slide technology such as double-label immunofluorescence microscopy to quantify nerve cell damage and loss of synaptic terminals in regions of increased iron;and electron microscopy to reveal cytosolic and mitochondrial ferritin. High normal iron concentration in a given tissue, such as the dentate nucleus, does not necessarily convey increased vulnerability to FRDA. Therefore, the globus pallidus with its equally high iron and ferritin levels will be added as an internal control. Furthermore, the neuropathological phenotype of some spinocerebellar ataxias (SCA) includes lesions of DRG. Samples of SCA will be studied in a parallel effort to determine that the changes in DRG of FRDA patients are specific for inherited frataxin deficiency. The work is clinically relevant because it will resolve questions about iron in the formal pathogenesis and natural history of FRDA, and the potential value of iron chelation. PUBLIC HEALTH RELEVANCE: Friedreich's ataxia (FRDA) is the most common hereditary ataxia, affecting 5,000-6,000 children and young adults in the United States at any given time. Carrier frequency is high at 1:50 to 1:100. The disease is due to a mutation in the frataxin gene, which controls important steps in energy and iron metabolism of all cells. Heart and peripheral and central nervous systems are most vulnerable. The mutation consists of an abnormal guanine-adenine-adenine trinucleotide repeat expansion, and persons with such an expansion on both parental alleles lack sufficient frataxin in their mitochondria. This research is devoted to the study of disturbed iron metabolism in the dentate nucleus of the cerebellum and dorsal root ganglia of the spinal cord because these anatomical structures are very susceptible to damage in FRDA. The research is expected to have high impact on our understanding of the natural history of FRDA and future therapy. Several clinical trials with experimental drugs are currently underway. The proposed research will provide the scientific basis for their effectiveness. The drug idebenone seeks to counteract iron-mediated oxidative damage and may be beneficial for the heart disease of patients with FRDA. Chelation therapy in an effort to remove iron from affected tissues remains controversial. The proposed investigation will help clarify the potential benefit of chelation therapy in FRDA.